Serotonin Receptor 2A −1438G/A Promoter Polymorphism in Relation to Obesity and Response to Sibutramine

Abstract

Aims: Serotonin has been related to appetite and body weight control. The aim of this study was to investigate a possible association of the −1438 G/A promoter polymorphism of the serotonin 2A receptor (5HT2AR) gene with obesity-related variables and response to sibutramine. Patients/Methods: We examined the potential impact of this polymorphism on obesity and related metabolic traits in a cohort of 234 overweight/obese and 103 lean Greek subjects. Additionally, we examined whether the 5HT2AR 1438A/G polymorphism influences weight reduction and change in body composition among 106 out of these subjects, who were treated with 15 mg sibutramine. Genotyping was carried out by polymerase chain reaction and restriction enzyme analysis. Results: Body mass index, fat mass, and waist circumference were not significantly different across the 5HT2AR 1438A/G genotype groups in overweight/obese women. Polymorphic G allele was associated with higher triglyceride and insulin levels but not with other biochemical and metabolic parameters. Distribution of genotypes and alleles was not different between responders and nonresponders (weight loss >5 or <5 kg). Conclusions: Based on these results, it seems unlikely that the 5HT2AR 1438 G/A polymorphism has a major impact on obesity and related traits or the response to sibutramine in Greek overweight/obese subjects.

Introduction

The fundamental causes of obesity relate to a complex interplay between environmental exposure, health behavior, and inherited components (Kopelman, 2000; Karam and McFarlane, 2010). Genetic factors are also thought to influence an individual's likelihood of becoming obese given a particular level of energy intake and expenditure (Costanzo and Schiffman, 1989; Hill and Peters, 1998).

The serotonergic system is important in the central nervous system regulation of many physiological processes including energy and glucose homeostasis (Halford et al., 2007; Gerozissis, 2008). Genetic variations that potentially affect serotonergic functions may influence appetite, glucose, and fat metabolism (Monteleone and Maj, 2008; Scherag et al., 2010). An MspI restriction fragment length polymorphism in the promoter region of the serotonin of the 2A receptor (5HT2AR −1438G/A) has been described (Collier et al., 1997). Contradictory data have been published for a possible association of this polymorphism with appetite and eating disorders (Collier et al., 1997; Aubert et al., 2000; Gorwood et al., 2002; Monteleone and Maj, 2008) or obesity and related metabolic features (Rosmond et al., 2002; Santos et al., 2005; Kring et al., 2009). Moreover, drugs that increase serotonergic transmission such as sibutramine, a centrally acting noradrenaline and serotonin reuptake inhibitor, enhance satiety and are used to support weight loss (Lean, 1997). However, weight loss in response to sibutramine is highly variable (James et al., 2000; Wirth and Krause, 2001). The reasons for the high variability in response to sibutramine are currently unknown.

Based upon the above considerations, the aim of the present study was to examine (i) the 5HT2AR −1438G/A polymorphism in relation to obesity and related metabolic traits and (ii) whether this common polymorphism modulates responder rates to sibutramine, among a cohort of Greek overweight/obese subjects.

Patients and Methods

To assess the association between 5HT2AR −1438G/A, obesity, and related metabolic traits, a total of 234 overweight/obese (body mass index [BMI], >25 kg/m²) adult subjects (94 males and 140 females; mean BMI=32.1±4.4 kg/m²) were studied. This study group was compared with a control group consisting of 103 unrelated age and sex-matched healthy lean individuals (43 males and 60 females; mean BMI=23.1±1.6 kg/m²). All obese subjects agreed to participate in a 3-month outpatient intervention program based on nutritional education, diet (500-kcal deficit), and encouragement for increasing physical activity. All patients had normal thyroid function and none of them had concomitant serious renal, hepatic, or cardiac disease. Antidiabetic agents or other drugs possibly inducing weight change were not added during the study period.

Sibutramine was subscribed (10 mg at first visit and 15 mg thereafter/day) in a total of 111 (out of the 234) overweight/obese subjects, according to the relative indications, product label, and the participants' willingness. Five patients dropped out of the study (because of noncompliance or undesirable side effects) so a total of 106 were available for the final evaluation. Based on body weight loss, subjects who received sibutramine were grouped as responders (weight loss >5% of their initial body weight, n=78) or nonresponders (weight loss <5% of their initial body weight, n=28).

All obese subjects were recruited from the Outpatient Clinic of Obesity, Diabetes, and Metabolism in the Second Department of Internal Medicine at Democritus University of Thrace. The study was conducted in accordance with the Helsinki Declaration of Human Rights and all subjects gave their informed consent.

Anthropometric measures and biochemical assays

Anthropometry was done with subjects wearing light, indoor clothing without shoes. The mean of two measurements was recorded for each variable. Body weight was measured to the nearest 0.1 kg on calibrated scales. Body height was measured with a calibrated stadiometer. Waist circumference was measured midway between the iliac crest and the lower costal margin, with the participant wearing only nonrestrictive underwear. Body composition was measured using the Skylark Body Fat analyzer (model BT-905). Arterial blood pressure was evaluated twice with the subject in the sitting position, 1 min apart. The mean value of both systolic and diastolic arterial pressure was considered. Biochemical measurements were performed in blood samples taken the morning after a 14-h fast, using standard enzymatic methods.

Genotyping

DNA was isolated from anticoagulated peripheral blood using a commercially available kit according to the manufacturer's instructions (QIAamp DNA Blood Mini Kit; Qiagen). Amplification of the 5HT2A gene containing the −1438G/A polymorphic site was carried out using standard polymerase chain reaction (PCR) techniques. The 468-bp amplification product was further digested using the endonuclease MspI. The PCR product for the G allele was cut into 224- and 244-bp fragments, whereas the A allele PCR product remained undigested.

Statistical analysis

Data are presented as the mean±standard deviation unless otherwise stated and p<0.05 was considered statistically significant. Each variable was checked for normal distribution using the Kolmogorov-Smirnov test and for homogeneity of variances using Levene's test. A χ² test was used to compare allelic frequencies between the groups. ANOVA was used to compare average values of anthropometric and biochemical parameters between genotypes. Differences among the responders' and nonresponders' groups were assessed by analysis of covariance (ANCOVA). Adjustments were made for the following covariates: sex, age, baseline weight, and presence or not of diabetes mellitus.

Results

Genotype frequencies were in Hardy-Weinberg equilibrium. The genotype and allele frequencies of the 5HT2AR −1438G/A polymorphisms were similar between the two groups (Table 1). BMI, fat mass, and waist circumference were not significantly different across the 5HT2AR 1438A/G genotype groups in overweight/obese subjects (Table 2). The polymorphic G allele was associated with higher triglycerides and insulin levels but not with other biochemical and metabolic parameters in the same group. A separate analysis based on the inclusion of women alone revealed similar results (data not shown).

Table 1.

Distribution Frequency of Genotypes and Alleles of 5HT2AR −1438G/A in Obese and Control Subjects

	Genotype			Allele
Group	AA (%)	A/G (%)	G/G (%)	A (%)	G (%)
Obese	60 (25.6%)	118 (50.4%)	56 (23.9%)	238 (50.8%)	230 (49.2%)
Lean	20 (19.4%)	56 (54.3%)	27 (26.2%)	96 (46.6%)	110 (53.4%)
p				0.31
OR				1.18
95% CI				0.85-1.64

OR, odds ratio; CI, confidence interval.

Table 2.

Clinical and Basic Metabolic Features of the Obese Participants According to Their Genotype

	Overweight/obesity group
	−1438A/A	−1438A/G	−1438G/G	p
BMI	32.2±4.7	31.7±3.9	33.2±4.3	0.32
Fat mass (%)	37.5±6.1	36.6±8.4	38.1±6.7	0.41
Waist (cm)	120±16	121±19	123±10	0.73
Fasting glucose (mg %)	113±49	117±55	115±41	0.65
Insulin (μIU/mL)	15.1±8.8	19.4±14.3	21.8±14.1	0.03
HOMA index	4.9±4.4	5.1±4.9	5.2±3.7	0.21
Total cholesterol (mg %)	208±43	217±67	210±34	0.60
LDL (mg %)	128±34	119±45	124±28	0.68
HDL (mg %)	53±11	50±14	47±13	0.19
Triglycerides (mg %)	135±68	170±75	175±89	0.04

BMI, body mass index; HOMA, Homeostasis Model Assessment; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Basic demographic features and anthropometric measurements at baseline (sex, age, BMI, fat mass, waist) were not statistically different between responders and nonresponders (data not shown). Distribution of genotypes and alleles was not different between responders and nonresponders. The A allele frequencies were 52.1% and 50.0% in the two groups, respectively (p=0.87, OR: 0.96, 95% CI: 0.50-1.77).

BMI loss after 3 months was 4.1±0.8, 4.3±1.4, and 4.0±1.0 kg/m² in the three genotypic groups, respectively. ANCOVA after adjustment for covariates (sex, age, baseline weight, and presence or not of diabetes mellitus) did not reveal a significant effect of the genotype on the response to sibutramine (F=1.1, p=0.45).

Discussion

We aimed to look for a possible association between 5HT2A receptor −1438G/A polymorphism and obesity and whether this common polymorphism modulates responder rates to sibutramine, among a cohort of Greek overweight/obese subjects. Our results suggest that the genetic polymorphism −1438A/G of the 5HT2AR is not associated with obesity or markers of obesity. The G allele was only found to be associated with higher insulin (but not glucose or HOMA index) and triglyceride levels. Our findings are in concordance with those of Hinney et al. (1997) and Kring et al. (2009) but in contrast with those published by Rosmond et al. (2002), Sorlí et al. (2008), and Ying et al. (2009). Interestingly, Zhang et al. (2010) showed that serotonin type 5A receptor sequence variants affect plasma levels of triglycerides in an obese cohort of families of Northern European descent. Discrepant results have been also published regarding a possible role of 5HT2AR gene in anorexia nervosa. A multicenter analysis, however, does not support a significant role of the 5HT2AR gene in anorexia nervosa (Gorwood et al., 2002).

It seems that the 5HT2AR −1438G/A polymorphism is highly unlikely to be associated with obesity in Greek individuals. The number of studied patients may have been too small to detect a weak genotype/phenotype association. Our study of quantitative traits, however, lend little support for a possible association between the G allele and obesity traits, given that no significant differences were demonstrated in BMI, fat mass, and waist circumference. Moreover, p-values in practically all investigated genotype/phenotype relationships were far from 0.05, so it is difficult to imagine that increasing the sample size would significantly change the results. The inconsistencies between association studies may reflect the complex interactions between multiple population-specific genetic and environmental factors. It is possible that minor genes that have been implicated in appetite and weight control, as in the case of the 5HT2AR gene, would have a relatively small effect on the development of obesity. If a causative relationship really exists, it would require time and the concurrence of other genetic and environmental factors for complete development.

Studies examining whether specific polymorphisms may influence weight loss and body composition change in response to sibutramine treatment amongst overweight/obese individuals are limited and a possible effect of 5HT2AR gene polymorphisms on weight loss in response to sibutramine treatment had not been studied so far. The distribution of genotypes and alleles in our study was not different between responders and nonresponders and it did reveal a significant effect of the 5-HT2AR genotypes on the response to sibutramine.

Grudell et al. (2008) in a very elegant study showed that treatment with sibutramine resulted in significantly greater reduction of body fat for specific genotype variants of the α2A and GNβ3 genes. The latter gene variant was also associated with a strong effect of sibutramine on weight reduction in obese Taiwanese patients (Hsiao et al., 2009). Several other candidate genes modifying noradrenaline metabolism or alpha2 adrenoreceptor function have also been investigated with various results (Camilleri, 2007; Kring et al., 2009). The general methodology for pharmacogenetic analysis using a gene-based approach like the one used in the present study looks for the association of single-nucleotide polymorphisms or haplotypes to explain the drug response. Identifying genetic markers in multiple genes in the drug response pathway seems more promising in unraveling a possible pharmacogenetic association.

In conclusion, our study showed that the 5HT2AR −1438G/A polymorphism is not related to obesity and major metabolic traits and does modulates responder rates to sibutramine, among a cohort of Greek overweight/obese subjects. In view of the epidemically increasing prevalence of obesity in most parts of the world and together with the fact that obesity is the most important risk factor for type 2 diabetes, effective nonpharmacological and pharmacological strategies to reduce body weight are urgently needed. Because of the limited success of lifestyle intervention programs, the very low number of currently available antiobesity drugs and the modest reductions in body weight they achieve, identifying those patients who are specifically responsive to a certain pharmacological agent is clearly worthwhile.

Footnotes

Disclosure Statement

The authors declare that they have no commercial associations that might create a conflict of interest in connection with this article.

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